Production of secreted polypeptides

ABSTRACT

Described herein are methods and compositions for the production and secretion of polypeptides. Included herein is the use of interrupting peptide transport activity for an increase in polypeptide production and/or secretion.

RELATED APPLICATIONS

The present application is a continuation to PCT/US99/31010 filed Dec.21, 1999, incorporated herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to the increased production of proteins,preferably, heterologous secreted proteins and cells having interruptedpeptide transport activity.

BACKGROUND

Secretion of heterologous polypeptides is a widely used technique inindustry. A cell can be transformed with a nucleic acid encoding aheterologous polypeptide of interest to be secreted and thereby producelarge quantities of desired polypeptides. This technique can be used toproduce a vast amount of polypeptide over what would be producednaturally. Polypeptides of interest have a number of industrialapplications, including therapeutic and agricultural uses, as well asuse in foods, cosmetics, cleaning compositions, animal feed, etc.

Thus, increasing secretion of polypeptides is of interest. Secretion ofpolypeptides into periplasmic space or into their culture media issubject to a variety of parameters. Typically, vectors for secretion ofa polypeptide of interest are engineered to position DNA encoding asecretory signal sequence 5′ to the DNA encoding the DNA of interest.

Attempts to increase secretion have often fallen into one of thefollowing three areas: trying several different signal sequences,mutating the signal sequence, and altering the secretory pathway withinthe host. While some success has been found with the above methods,generally, they are time consuming and novel methods are desirable.Therefore, a problem to be solved is how to produce and/or secrete moreproteins without solely relying on altering the signal sequence.

The instant invention provides a novel approach to improving secretionof polypeptides in a cell. Also provided herein are novel compositionsuseful in the methods of polypeptide secretion provided herein, andmethods of making such compositions.

SUMMARY OF THE INVENTION

In one aspect of the invention, a method of increasing secretion of apolypeptide in a cell is provided. In a preferred embodiment, said cellselected would express at least one peptide transport protein. In oneembodiment, the method comprises inactivating said at least one peptidetransport protein in said cell and culturing said cell under conditionssuitable for expression and secretion of said polypeptide.

The methods provided herein are applicable for production or secretionof polypeptides in a variety of cell types. For example, the cell can beselected from the group consisting of a plant cell, a fungal cell, agram-negative microorganism and a gram-positive microorganism. In oneembodiment, said cell is a gram-negative microorganism, preferably, amember of the family Escherichia. In another embodiment, said cell is agram-positive microorganism, preferably a member of the family Bacillus.In a preferred embodiment, said cell is a gram-positive microorganismand is a member of the family Bacillus wherein said member of the familyBacillus is selected from the group consisting of B. licheniformis, B.lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B.amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B.thuringiensis, B. methanolicus and B. anthracis.

The polypeptide which is secreted or produced by the methods providedherein can be any polypeptide. In a preferred embodiment, it is aheterologous polypeptide. In one aspect, the polypeptide is selectedfrom the group consisting of hormone, enzyme, growth factor andcytokine. In one embodiment, the polypeptide is an enzyme, preferablyselected from the group consisting of proteases, carbohydrases,reductases, lipases, isomerases, transferases, kinases, phophatases,cellulase, endo-glucosidase H, oxidase, alpha-amylase, glucoamylase,lignocellulose hemicellulase, pectinase and ligninase. In one aspect,said polypeptide is a bacillus protease, preferably subtilisin. Inanother aspect, said polypeptide is an amylase, preferably, bacillusamylase.

The peptide transport protein can be a variety of proteins and can beinactivated in a variety of ways. In one aspect, the peptide transportprotein is a gene product of a dciA operon, and preferably, is the geneproduct of the dciAE gene. The protein can be inactivated at the proteinor nucleic acid level. In one aspect, the protein is inactivated becausethe gene encoding said protein has been mutated. In another embodiment,the operon comprising said gene has been mutated. The mutation can becaused in a variety of ways including one or more frameshifts,insertions, substitutions and deletions, or combinations thereof. Thedeletion can be of a single nucleotide or more, including deletion ofthe entire gene.

In another aspect of the invention, a method for producing a polypeptidein a cell is provided which comprises the steps of obtaining a cellcomprising nucleic acid encoding a polypeptide to be produced, said cellfurther comprising a peptide transport operon wherein at least one geneproduct of said operon is inactive in said cell, and culturing said cellunder conditions suitable for expression such that said polypeptide isproduced. Preferably, the peptide transport operon is a dciA operon. Thegene product of said operon can be inactivated at the nucleic acid orprotein level. Preferably, the inactivated gene product is encoded bydciAA or dciAE.

In another aspect of the invention provided herein is a cell comprisinga peptide transport operon, wherein said operon has been mutated suchthat said cell has increased polypeptide secretion. In a preferredembodiment, said operon is a dciA operon. In one embodiment, said operonhas been mutated to inactivate a gene product of a dciAA and/or dciAEgene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show an embodiment of a nucleic acid (SEQ ID NO:1)encoding a dciA operon, wherein the nucleotides encoding dciAA and dciAEare shown, respectively, underlined and in bold.

FIG. 2 shows an embodiment of an amino acid sequence for dciAA (SEQ IDNO: 2), encoded by nucleotides 210-1034 of the nucleic acid sequenceshown in FIGS. 1A and 1B, wherein the amino acids shown in underlinedare omitted in an embodiment of inactive dciAA provided herein.

FIG. 3 shows an embodiment of an amino acid sequence for dciAB (SEQ IDNO: 3), encoded by nucleotides 1051-1977 of the nucleic acid sequenceshown in FIGS. 1A and 1B.

FIG. 4 shows an embodiment of an amino acid sequence for dciAC (SEQ IDNO: 4), encoded by nucleotides 1983-2945 of the nucleic acid sequenceshown in FIGS. 1A and 1B.

FIG. 5 shows an embodiment of an amino acid sequence for dciAD (SEQ IDNO: 5), encoded by nucleotides 2950-3957 of the nucleic acid sequenceshown in FIGS. 1A and 1B.

FIG. 6 shows an embodiment of an amino acid sequence for dciAE (SEQ IDNO: 6), encoded by nucleotides 3978-5609 of the nucleic acid sequenceshown in FIGS. 1A and 1B, wherein the amino acids shown in underlinedare omitted in an embodiment of inactive dciAA provided herein.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of the invention, a method of increasing production and/orsecretion of a polypeptide in a cell is provided. In a preferredembodiment, said cell selected for said method expresses at least onepeptide transport protein. Preferably, the cell selected endogenouslyexpresses said peptide transport protein, however, the cell can betransformed to express said protein. In one embodiment, the methodcomprises inactivating at least one peptide transport protein in saidcell. The method further comprises culturing said cell under conditionssuitable for expression and secretion of said polypeptide.

“Peptide transport protein” as used herein refers to a protein involvedin peptide transport. Proteins involved in peptide transport areconsidered to have peptide transport activity. In one embodiment, apeptide transport protein includes a protein involved in dipeptide oroligopeptide transport, preferably dipeptide transport. In oneembodiment, the peptides which are transported by the peptide transportproteins or systems described herein include peptides which are 2-10amino acids long, preferably 2-8, and more preferably 2-6. In apreferred embodiment, oligopeptides are 3-7 amino acids long, preferably5 or 6 amino acids long and dipeptides are two amino acids.

The peptide transport protein which is inactivated is preferablyinvolved in the import of proteins. As further discussed below,inactivation can occur in a variety of ways and by individual mutationsor combinations of mutations. Peptide transport systems can includeactivity in exporting and/or importing peptides. In a preferredembodiment herein, peptide transport activity is interrupted.Preferably, importation of peptides by a peptide transport system isdecreased or eliminated. Peptide transport proteins are known in the artand include proteins or gene products encoded by peptide transportoperons as discussed below and known in the art.

An “operon” as used herein refers to a cluster of genes that are allcontrolled by the same promoter. A peptide transport operon includes atleast one gene encoding a peptide transport protein. Peptide transportoperons include the opp operon and the dciA operon and homologs thereof.In one embodiment herein, a peptide transport operon excludes the oppoperon. In another embodiment herein, a peptide transport proteinexcludes proteins encoded by the opp operon.

The opp operon has been reported as encoding an oligopeptide permeasethat is required for the initiation of sporulation and the developmentof genetic competence (Rudner et al, 1991, Journal of Bacteriology,173:1388-1398). The opp operon is a member of the family of ATP-bindingcassette transporters involved in the import or export of oligopeptidesfrom 3-5 amino acids. There are five gene products of the opp operon:oppA is the ligand-binding protein and is attached to the outside of thecell by a lipid anchor; oppB and oppC are the membrane proteins thatform a complex through which the ligand is transported; oppD and oppF(Perego et al., 1991, Mol. Microbiol. 5:173-185) are the ATPases thoughtto provide energy for transport (LeDeaux, J. R., et al., 1997, FEMSMicrobiology Letters 153: 63-69). The opp operon has also been referredto as SpoOK by Rudner et al., 1991, J. Bacteriol. 173:1388-1398.

Opp operons are also disclosed in Podbielski et al. 1996, MolecularMicrobiology 21: 1087-1099 and Tynkkynen et al. 1993, Journal ofBacteriology 175: 7523-7532. One assay for the presence or absence of afunctioning opp operon is to subject the host to growth in the presenceof toxic oligopeptide of 3 amino acids, such as Bialaphos, a tripeptideconsisting of two L-alanine molecules and an L-glutamic acid analogue(Meiji Seika, Japan). A cell having a functional opp operon will haveinhibited growth. A cell having a mutation in at least one gene of theopp operon gene cluster will not show growth inhibition in the presenceof the toxic oligopeptide.

Further regarding peptide transport operons and peptides thereof, oppA,B,C,D, F of Salmonella typhimurium are further reported on in Hiles etal.,1987 J. Mol. Biol 195:125-142. OppA, B, C, D, F of Bacillus subtilisare further reported on in Perego, M., et al. Mol Microbiol 1991, 5,173-185 and Rudner, D. Z., et al., J. Bacteriol 1991, 173(4):1388-1398.OppA, B, C, D, E of E. coli are reported on in Guyer, et al., 1985 J.Biol Chem 260:10812-10816. Also, a report has been made on amiA ofStreptococcus pneumonia (Alloing, G 1994, J Mol Biol 241(1) :44-58.AppA, B, C, D, E of B. subtilis are reported on in Koide A. et al MolMicrobiol 1994 13(3) :417-426. Additionally, dppA of E. coli and S.typhimurium is reported on in Abouhamad et al 1991 Mol Microbiol5(5):1035-1047. Furthermore, BldA, B, C, D, E of Streptomyces coelicolorare reported on in Nodwell, J. R. et al Mol Microbiol 1996,22(5):881-93. OppA of Streptococcus is reported on in Podbielski, A etal Mol Microbiol 1996, 21(5):1087-1099. Moreover, TppA, B, C, D, E of S.typhimurium have been reported on in Gibson, M. M. et al., J. Bacteriol1984 160:122-130. Moreover, it has been reported that oppA may also beobtained from Chlamydia pneumonia.

Organisms which a peptide transport operon or protein thereof can beobtained from include but are not limited to: Aquifex aeolicus,Archaeoglobus fulgidus, Aeropyrum pernix, Bordetella pertussis, Bacillussubtilis, Clostridium acetobutylicum, Campylobacter jejuni, Chlorobiumtepidum, Chlamydia pneumoniae CWL029, Chlamydia trachomatis Serovar D,Clostridium difficile, Corynebacterium diphtheriae, Deinococusradiodurans, Escherichia coli, Enterococcus faecalis, Haemophilusinfluenzae, Helicobacter pylori, Klebsiella pneumoniae, Mycobacteriumleprae, Pseudomonas aeruginosa, Pyrococcus furiosus, Pyrococcushorikoshii, Pyrococcus abysii, Rhodobacter capsulatus, Streptococcuspyogenes and Salmonella typhimurium.

The dciA operon has been reported on, see, e.g., Slack, et al., MolMicrobio (1991) 5(8), 1915-1925 and Mathlopoulos, et al., Mol Microbio(1991) 5(8), 1903-1913, as being a dipeptide transport operon inBacillus. While this operon has been reported on, the function of eachgene product has not previously been well characterized. Herein,provided are functions of the proteins of the dciA operon, includingfunctional properties wherein a gene product is inactivated.

In one embodiment, dciAA is a peptide transport protein and inactivationof dciAA leads to increased polypeptide production and/or secretion. Inanother embodiment, inactivation of dciAE leads to decreased peptidetransport and increased polypeptide production and/or secretion. Inpreferred embodiments, dciAA has RNA binding activity. Embodiments ofdciAA, dciAB, dciAC, dciAD and dciAE are shown in FIGS. 2-6,respectively. DciA has homology to dpp of E. coli. Olson, et al., JBacteriol 173:234-244 (1991); Kawarabayasi, Y., Journal DNA Res. 5 (2),55-76 (1998).

In addition to those described above, homologs of peptide transportoperons, peptide transport genes and peptide transport proteins can beidentified by a number of methods. In one embodiment, a nucleic acid isa “peptide transport gene” if it encodes a protein having peptidetransport activity as discussed above. Preferably, the overall homologyof the nucleic acid sequence is preferably greater than about 60%, morepreferably greater than about 75%, more preferably greater than about80%, even more preferably greater than about 85% and most preferablygreater than 90% of one of the dciA genes shown in FIG. 1, namely dciAA,dciAB, dciAC, dciAD or dciAE. In some embodiments the homology will beas high as about 93 to 95 or 98%.

In one embodiment, a protein is a “peptide transport protein” if it haspeptide transport activity. Preferably the protein has overall homologygreater than about 40%, more preferably greater than about 60%, morepreferably at least 75%, more preferably greater than about 80%, evenmore preferably greater than about 85% and most preferably greater than90% to the amino acid sequence of FIGS. 2, 3, 4, 5, or 6. In someembodiments the homology will be as high as about 93 to 95 or 98%.

Homology as used herein is in reference to sequence similarity oridentity, with identity being preferred. This homology will bedetermined using standard techniques known in the art, including, butnot limited to, the local homology algorithm of Smith & Waterman, Adv.Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman & Wunsch, J. Mol. Biool. 48:443 (1970), by the search forsimilarity method of Pearson & Lipman, PNAS USA 85:2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Drive, Madison, Wis.), the Best Fit sequence programdescribed by Devereux et al., Nucl. Acid Res. 12:387-395 (1984),preferably using the default settings, or by inspection.

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987); the method is similar to that described by Higgins &Sharp CABIOS 5:151-153 (1989). Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin etal., PNAS USA 90:5873-5787 (1993). A particularly useful BLAST programis the WU-BLAST-2 program which was obtained from Altschul et al.,Methods in Enzymology, 266: 460480 (1996). WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2parameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity. A % amino acid sequence identity value isdetermined by the number of matching identical residues divided by thetotal number of residues of the “longer” sequence in the aligned region.The “longer” sequence is the one having the most actual residues in thealigned region (gaps introduced by WU-Blast-2 to maximize the alignmentscore are ignored).

Thus, “percent (%) nucleic acid sequence identity” is defined as thepercentage of nucleotide residues in a candidate sequence that areidentical with the nucleotide residues of the sequence shown in thenucleic acid figures. A preferred method utilizes the BLASTN module ofWU-BLAST-2 set to the default parameters, with overlap span and overlapfraction set to 1 and 0.125, respectively.

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer nucleosides than those of the nucleic acid figures, it isunderstood that the percentage of homology will be determined based onthe number of homologous nucleosides in relation to the total number ofnucleosides. Thus, for example, homology of sequences shorter than thoseof the sequences identified herein and as discussed below, will bedetermined using the number of nucleosides in the shorter sequence.

In one embodiment, the peptide transport gene or operon is determinedthrough hybridization studies. Thus, for example, nucleic acids whichhybridize under high stringency to the nucleic acid sequences (that ofthe operon, the individual genes thereof or fragments thereof)identified in the figures, or a complement, are considered a peptidetransport gene in one embodiment herein. High stringency conditions areknown in the art; see for example Maniatis et al., Molecular Cloning: ALaboratory Manual, 2d Edition, 1989, and Short Protocols in MolecularBiology, ed. Ausubel, et al., both of which are hereby incorporated byreference. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, “Overview of principles of hybridization and the strategy ofnucleic acid assays” (1993). Generally, stringent conditions areselected to be about 5-10 C. lower than the thermal melting point (Tm)for the specific sequence at a defined ionic strength pH. The Tm is thetemperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at Tm, 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g. 10 to 50nucleotides) and at least about 60° C. for long probes (e.g. greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

In another embodiment, less stringent hybridization conditions are used;for example, moderate or low stringency conditions may be used, as areknown in the art; see Maniatis and Ausubel, supra, and Tijssen, supra.

Naturally occuring allelic variants of the genes and proteins providedherein may also be used in the methods of the present invention.

In addition to using the above techniques to find homologs of peptidetransport genes, operons, proteins or peptide transport operon genes andproteins, one may use standard amplification of fragments of sequencesas provided herein carried out in polymerase chain reaction (PCR)technologies such as described in Dieffenbach CW and GS Dveksler (1995,PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, PlainviewN.Y.). A nucleic acid sequence of at least about 10 nucleotides and asmany as about 60 nucleotides from an operon gene as provided herein,preferably about 12 to 30 nucleotides, and more preferably about 20-25nucleotides can be used as a probe or PCR primer.

The term “nucleic acid” as used herein refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences and as wellas the sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Cassol et al., 1992; Rossolini et al., Mol. Cell. Probes 8:91-98(1994)). The term nucleic acid is used interchangeably with gene, cDNA,and mRNA encoded by a gene.

“Protein” as used herein includes proteins, polypeptides, and peptides.As will be appreciated by those in the art, the nucleic acid sequencesof the invention can be used to generate protein sequences. Preferredpeptide transport proteins have peptide transport activity prior toinactivation and/or are controlled by a peptide transport operon.

The methods provided herein are applicable for production or secretionof polypeptides in a variety of cell types including eukaryote andprokaryote. For example, the cell can be selected from the groupconsisting of a plant cell, a mammalian cell, an insect cell, fungalcell, a gram-negative microorganism and a gram-positive microorganism.

Fungal cell or fungi as used herein include Chytridiomycetes,Hyphochrytridiomycetes, Plasmodiophoromycetes, Oomycetes, Zygomycetes,Trichomycetes, Ascomycetes, and Basidiomycetes. In one embodiment,filamentous fungi are used. Various species of filamentous fungi may beused as expression hosts including from the following genera:Aspergillus, Trichoderma, Neurospora, Penicillium, Cephalosporium,Achlya, Phanerochaete, Podospora, Endothia, Mucor, Fusarium, Humicola,Cochliobolus and Pyricularia. One embodiment includes Penicilliumchrysogenum. One embodiment includes Fusarium solani. Specificexpression hosts include A. nidulans, (Yelton, M., et al. (1984) Proc.Natl. Acad. Sci. USA, 81, 1470-1474; Mullaney, E. J. et al. (1985) Mol.Gen. Genet. 199, 37-45; John, M. A. and J. F. Peberdy (1984) EnzymeMicrob. Technol. 6, 386-389; Tilburn, et al. (1982) Gene 26, 205-221;Ballance, D. J. et al., (1983) Biochem. Biophys. Res. Comm. 112,284-289; Johnston, I. L. et al. (1985) EMBO J. 4, 1307-1311) A. niger,(Kelly, J. M. and M. Hynes (1985) EMBO 4, 475-479) A. awamori, e.g.,NRRL 3112, ATCC 22342, ATCC 44733, ATCC 14331 and strain UVK 143f, A.oryzae, e.g., ATCC 11490, N. crassa (Case, M. E. et al. (1979) Proc.Natl. Acad. Scie. USA 76, 5259-5263; Lambowitz U.S. Pat. No. 4,486,553;Kinsey, J. A. and J. A. Rambosek (1984) Molecular and Cellular Biology4, 117-122; Bull, J. H. and J. C. Wooton (1984) Nature 310, 701-704),Trichoderma reesei, e.g. NRRL 15709, ATCC 13631, 56764, 56765, 56466,56767, and Trichoderma viride, e.g., ATCC 32098 and 32086.

In another embodiment, yeast cells are utilized or provided herein forproduction or secretion of polypeptides. Yeasts are suitable herein andinclude, but are not limited to, yeast capable of growth on methanolselected from the genera consisting of Hansenula, Candida, Kloeckera,Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specificspecies that are exemplary of this class of yeasts may be found in C.Anthony, The Biochemistry of Methylotrophs, 269 (1982). For example,Candida species includes but is not limited to Candida albicans, Candidatropicalis, Candida (Torulopsis) glabrata, Candida parapsilosis, Candidalusitaneae, Candida rugosa and Candida pseudotropicalis.

In one embodiment, the cell utilized or provided herein is agram-negative microorganism. In one embodiment, the cell is fromEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, Shigella or Pseudomonas such as P.aeruginosa.

In another embodiment, said cell is a gram-positive microorganism,preferably a member of the family Bacillus, although other gram-positivecells can be used such as those from Streptomyces. In a preferredembodiment, said cell is a gram-positive microorganism and is a memberof the family Bacillus wherein said member of the family Bacillus isselected from the group consisting of B. licheniformis, B. lentus, B.brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B.coagulans, B. circulans, B. lautus, B. thuringiensis, B. methanolicusand B. anthracis.

The polypeptide which is secreted or produced by the methods providedherein can be any polypeptide of interest. In a preferred embodiment, itis a heterologous polypeptide. Alternatively, the protein is homologousas discussed below.

In one aspect, the polypeptide to be produced and/or secreted isselected from the group consisting of hormone, enzyme, growth factor andcytokine. In one embodiment, the polypeptide is an enzyme. An enzyme asused herein includes but is not limited to (i) oxidoreductases; (ii)transferases, comprising transferase transferring one-carbon groups(e.g., methyltransferases, hydroxymethyl-, formyl-, and relatedtransferases, carboxyl- and carbamoyltransferases, amidinotransferases)transferases transferring aldehydic or ketonic residues,acyltransferases (e.g., acyltransferases, aminoacyltransferas),glycosyltransferases (e.g., hexosyltransferases, pentosyltransferases),transferases transferring alkyl or related groups, transferasestransferring nitrogenous groups (e.g., aminotransferases,oximinotransferases), transferases transferring phosphorus-containinggroups (e.g., phosphotransferases, pyrophosphotransferases,nucleotidyltransferases), transferases transferring sulfur-containinggroups (e.g., sulfurtransferases, sulfotransferases, CoA-transferases),(iii) Hydrolases comprising hydrolases acting on ester bonds (e.g.,carboxylic ester hydrolases, thioester hydrolases, phosphoric monoesterhydrolases, phosphoric diester hydrolases, triphosphoric monoesterhydrolases, sulfuric ester hydrolases), hydrolases acting on glycosylcompounds (e.g., glycoside hydrolases, hydrolyzing N-glycosyl compounds,hydrolyzing S-glycosyl compound), hydrolases acting on ether bonds(e.g., thioether hydrolases), hydrolases acting on peptide bonds (e.g.,aminoacyl-peptide hydrolases, peptidyl-amino acid hydrolases, dipeptidehydrolases, peptidyl-peptide hydrolases), hydrolases acting on C—N bondsother than peptide bonds, hydrolases acting on acid-anhydride bonds,hydrolases acting on C—C bonds, hydrolases acting on halide bonds,hydrolases acting on P—N bonds, (iv) lyases comprising carbon-carbonlyases (e.g., carboxy-lyases, aldehyde-lyases, ketoacid-lyases),carbon-oxygen lyases (e.g., hydro-lyases, other carbon-oxygen lyases),carbon-nitrogen lyases (e.g., ammonia-lyases, amidine-lyases),carbon-sulfur lyases, carbon-halide lyases, other lyases, (v) isomerasescomprising racemases and epimerases, cis-trans isomerases,intramolecular oxidoreductases, intramolecular transferases,intramolecular lyases, other isomerases, (vi) ligases or synthetasescomprising ligases or synthetases forming C—O bonds, forming C—S bonds,forming C—N bonds, forming C—C bonds.

The polypeptide of interest may be a therapeutically significantprotein, such as growth factors, cytokines, ligands, receptors andinhibitors, as well as vaccines and antibodies.

In a preferred embodiment, the precursor mRNA for the polypeptide to beproduced or secreted includes a putative RNase cleavage site or abinding site for a peptide transport protein. In preferred embodiments,the polypeptide to be produced or secreted includes a specific peptidetransport protein recognition site. Preferably the recognition site is abinding site. Preferably, the recognition site is a binding site fordciAA.

As used herein, the term “heterologous protein” refers to a protein orpolypeptide that does not naturally occur in the host cell. The term“homologous protein” or “endogenously expressed” refers to a protein orpolypeptide native or naturally occurring in the host cell. In oneembodiment, the invention includes host cells producing the homologousprotein via recombinant DNA technology. A recombinant protein refers toany protein encoded by a nucleic acid which has been introduced into thehost.

“Inactive protein” or grammatical equivalents as used herein refers to areduction in the detectable activity of the protein when expressed inits wildtype form such as in its native unaltered host. Activities ofpeptide transport proteins include transport of dipeptides oroligopeptides of 3-7 amino acids, more preferably 5 or 6 amino acids,preferably in association with importation into the cell. In a preferredembodiment, peptide transport activity is reduced or eliminated.

In one aspect of the invention a gene product of a peptide transportoperon is inactive or inactivated when the operon is activated.Preferably, the inactivated gene product is encoded by dciAA and/ordciAE. In a preferred embodiment, wildtype gene product corresponding tothe inactive gene product comprises at least RNA binding or Rnaseactivity wherein the inactive gene product has decreased or eliminatedinteraction with RNA.

A peptide transport protein or a gene product of a peptide transportoperon can be inactivated at the protein or nucleic acid level. It isunderstood that the cells and methods of the present invention mayinclude more than one inactive protein. In one embodiment, two operonsare utilized, an opp operon and a dciA operon. In one embodiment, atleast one gene product from an opp operon and at least one gene productfrom a dciA operon is inactive.

While a number of examples are discussed herein, it is understood thatinactivation can occur by one or more mutations or modifications in agene, protein or operon, or a combination thereof. For example, in oneembodiment, a mutation is made in the oppA operon and one mutation ismade in the dciA operon, and more particularly in oppAA and dciAA. Inanother embodiment, 2 or more mutations are made to the same gene,protein or operon. In another embodiment, 2 or more mutations are madewherein the mutations occur in different genes of the same operon, or indifferent operons, wherein the mutations can be in the same gene ordifferent genes when the mutations are made in different operons.Generally, as used is herein, mutation is used interchangeably withmodification to refer to a change which infers inactivation of the gene,protein, system or activity as described herein.

In one aspect, the protein is inactivated because the gene encoding saidprotein has been mutated. The mutation can be caused in a variety ofways including one or more frameshifts, substitutions, insertions and/ordeletions as further described below. The deletion can be of a singlenucleotide or more, including deletion of the entire gene. It isunderstood that the cells comprising the inactive proteins describedherein and methods of making said cells are also provided herein.

In one embodiment, a cell having an inactive protein as described hereinis arrived at by the replacement and/or inactivation of the naturallyoccurring gene from the genome of the host cell. In a preferredembodiment, the mutation is a non-reverting mutation.

One method for mutating nucleic acid encoding a gene is to clone thenucleic acid or part thereof, modify the nucleic acid by site directedmutagenesis and reintroduce the mutated nucleic acid into the cell on aplasmid. By homologous recombination, the mutated gene may be introducedinto the chromosome. In the parent host cell, the result is that thenaturally occurring nucleic acid and the mutated nucleic acid arelocated in tandem on the chromosome. After a second recombination, themodified sequence is left in the chromosome having thereby effectivelyintroduced the mutation into the chromosomal gene for progeny of theparent host cell.

Another method for inactivating the gene product is through deleting thechromosomal gene copy. In a preferred embodiment, the entire gene isdeleted, the deletion occurring in such as way as to make reversionimpossible. In another preferred embodiment, a partial deletion isproduced, provided that the nucleic acid sequence left in the chromosomeis too short for homologous recombination with a plasmid encoded gene.

Deletion of the naturally occurring gene can be carried out as follows.A gene including its 5′ and 3′ regions is isolated and inserted into acloning vector. The coding region of the gene is deleted form the vectorin vitro, leaving behind a sufficient amount of the 5′ and 3′ flankingsequences to provide for homologous recombination with the naturallyoccurring gene in the parent host cell. The vector is then transformedinto the host cell. The vector integrates into the chromosome viahomologous recombination in the flanking regions. This method leads to astrain in which the gene has been deleted.

The vector used in an integration method is preferably a plasmid. Aselectable marker may be included to allow for ease of identification ofdesired recombinant microorgansims. Additionally, as will be appreciatedby one of skill in the art, the vector is preferably one which can beselectively integrated into the chromosome. This can be achieved byintroducing an inducible origin of replication, for example, atemperature sensitive origin into the plasmid. By growing thetransformants at a temperature to which the origin of replication issensitive, the replication function of the plasmid is inactivated,thereby providing a means for selection of chromosomal integrants.Integrants may be selected for growth at high temperatures in thepresence of the selectable marker, such as an antibiotic. Integrationmechanisms are described in WO 88/06623.

Integration by the Campbell-type mechanism can take place in the 5′flanking region of the gene, resulting in a strain carrying the entireplasmid vector in the chromosome in the locus. Since illegitimaterecombination will give different results it will be necessary todetermine whether the complete gene has been deleted, such as throughnucleic acid sequencing or restriction maps.

Another method of inactivating the naturally occurring gene is tomutagenize the chromosomal gene copy by transforming a cell witholigonucleotides which are mutagenic. Alternatively, the chromosomalprotease gene can be replaced with a mutant gene by homologousrecombination.

In a preferred embodiment, the present invention encompasses host cellshaving further protease deletions or mutations. For example, well knowprotease deletions in Bacillus, including deletions or mutations in apr,npr, epr, mpr generally used for efficient heterologous expression.Other embodiments of the protease are described in, for example, U.S.Pat. No. 5,264,366.

Other ways of inactivating a protein at the nucleic acid level includethe use of antisense molecules. Antisense or sense oligonucleotides,according to the present invention, comprise a fragment of the codingregion of a peptide transport protein or a product of a peptidetransport operon. Such a fragment generally comprises at least about 14nucleotides, preferably from about 14 to nucleotides. The ability toderive an antisense or a sense oligonucleotide, based upon a cDNAsequence encoding a given protein is described in, for example, Steinand Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al.(BioTechniques 6:958, 1988). Binding of antisense or senseoligonucleotides to target nucleic acid sequences results in theformation of duplexes that block transcription or translation of thetarget sequence by one of several means, including enhanced degradationof the duplexes, premature termination of transcription or translation,or by other means.

Ribozymes may also be used for inactivation in one embodiment. Ribozymesare enzymatic RNA molecules capable of catalyzing the specific cleavageof RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology, 4:469-471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

The peptide transport protein or product of a peptide transport operonmay also be inactive or inactivated at the protein level. For example,the nucleic acid encoding the protein or gene product may be intact, butthe cell may comprise an antagonist or inhibitor such as an antibody toinhibit the protein or gene product from having its native activity,such as peptide transport or RNA interaction activity. Moreover, theprotein may be expressed as an inactive variant or be conditionallyinactive, for example, by having temperature sensitive peptidetransport.

For production and/or secretion of proteins in a cell, an expressionvector comprising at least one copy of a nucleic acid encoding theheterologous or homologous protein, and preferably comprising multiplecopies, is transformed into the cell under conditions suitable forexpression of the protein.

Expression vectors used in the present invention comprise at least onepromoter associated with the protein, which promoter is functional inthe host cell. In one embodiment of the present invention, the promoteris the wild-type promoter for the selected protein and in anotherembodiment of the present invention, the promoter is heterologous to theprotein, but still functional in the host cell. In one preferredembodiment of the present invention, nucleic acid encoding thepolypeptide of interest is stably integrated into the host genome.Signal sequences may be added if needed.

In a preferred embodiment, the expression vector contains a multiplecloning site cassette which preferably comprises at least onerestriction endonuclease site unique to the vector, to facilitate easeof nucleic acid manipulation. In a preferred embodiment, the vector alsocomprises one or more selectable markers. As used herein, the termselectable marker refers to a gene capable of expression in the hostwhich allows for ease of selection of those hosts containing the vector.Examples of such selectable markers include but are not limited toantibiotics, such as, erythromycin, actinomycin, chloramphenicol andtetracycline.

In one embodiment of the present invention, nucleic acid encoding atleast one polypeptide of interest is introduced into a host cell via anexpression vector capable of replicating within the host cell. Suitablereplicating plasmids for Bacillus are described in Molecular BiologicalMethods for Bacillus, Ed. Harwood and Cutting, John Wiley & Sons, 1990,hereby expressly incorporated by reference; see chapter 3 on plasmids.Suitable replicating plasmids for B. subtilis are listed on page 92.Several strategies have been described in the literature for the directcloning of DNA in Bacillus. Plasmid marker rescue transformationinvolves the uptake of a donor plasmid by competent cells carrying apartially homologous resident plasmid (Contente et al., Plasmid2:555-571 (1979); Haima et al., Mol. Gen. Genet. 223:185-191 (1990);Weinrauch et al., J. Bacteriol. 154(3):1077-1087 (1983); and Weinrauchet al., J. Bacteriol. 169(3):1205-1211 (1987)). The incoming donorplasmid recombines with the homologous region of the resident “helper”plasmid in a process that mimics chromosomal transformation.

Transformation by protoplast transformation is described for B. subtilisin Chang and Cohen, (1979) Mol. Gen. Genet 168:111-115; for B.megateriumin Vorobjeva et al., (1980) FEMS Microbiol. Letters 7:261-263; for B.amyloliquefaciens in Smith et al., (1986) Appl. and Env. Microbiol.51:634; for B.thuringiensis in Fisher et al., (1981) Arch. Microbiol.139:213-217; for B.sphaericus in McDonald (1984) J. Gen. Microbiol.130:203; and B.larvae in Bakhiet et al., (1985) 49:577. Mann et al.,(1986, Current Microbiol. 13:131-135) report on transformation ofBacillus protoplasts and Holubova, (1985) Folia Microbiol. 30:97)disclose methods for introducing DNA into protoplasts using DNAcontaining liposomes. The presence/absence of a marker gene can suggestwhether the gene of interest is present in the host cell.

Alternatively, host cells which contain the coding sequence for thepolypeptide of interest may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay orimmunoassay techniques which include membrane-based, solution-based, orchip-based technologies for the detection and/or quantification of thenucleic acid or protein.

There are various assays known to those of skill in the art fordetecting and measuring activity of secreted polypeptides. Inparticular, for proteases, there are assays based upon the release ofacid-soluble peptides from casein or hemoglobin measured as absorbanceat 280 nm or calorimetrically using the Folin method (Bergmeyer, et al.,1984, Methods of Enzymatic Analysis vol. 5, Peptidases, Proteinases andtheir Inhibitors, Verlag Chemie, Weinheim). Other assays involve thesolubilization of chromogenic substrates (Ward, 1983, Proteinases, inMicrobial Enzymes and Biotechnology (W. M. Fogarty, ed.), AppliedScience, London, pp. 251-317).

Means for determining the levels of secretion of a heterologous orhomologous protein in a host cell and detecting secreted proteinsinclude, using either polyclonal or monoclonal antibodies specific forthe protein. Examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).These and other assays are described, among other places, in Hampton Ret al (1990, Serological Methods, a Laboratory Manual, APS Press, StPaul Minn.) and Maddox D E et al (1983, J Exp Med 158:1211). In apreferred embodiment, secretion is higher using the methods andcompositions provided herein than when using the same methods orcompositions, but where a peptide transport protein or gene product of apeptide transport operon has not been inactivated. In a preferredembodiment, wherein RNase activity is decreased, production and/orsecretion of polypeptides is increased. In another preferred embodiment,wherein peptide transport activity is decreased, production and/orsecretion is increased.

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic and amino acidassays. Means for producing labeled hybridization or PCR probes fordetecting specific polynucleotide sequences include oligolabeling, nicktranslation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the nucleotide sequence, or any portion ofit, may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3 or SP6 and labeled nucleotides.

A number of companies such as Pharmacia Biotech (Piscataway N.J.),Promega (Madison Wis.), and US Biochemical Corp (Cleveland Ohio) supplycommercial kits and protocols for these procedures. Suitable reportermolecules or labels include those radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles and the like. Patents teachingthe use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also,recombinant immunoglobulins may be produced as shown in U.S. Pat. No.4,816,567 and incorporated herein by reference.

The cells transformed with polynucleotide sequences encodingheterologous or homologous protein or endogenously having said proteinmay be cultured under conditions suitable for the expression andrecovery of the encoded protein from cell culture. Other recombinantconstructions may join the heterologous or homologous polynucleotidesequences to nucleotide sequence encoding a polypeptide domain whichwill facilitate purification of soluble proteins (Kroll D J et al (1993)DNA Cell Biol 12:441-53).

Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals (Porath J (1992) Protein Expr Purif3:263-281), protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle Wash.). The inclusion of acleavable linker sequence such as Factor XA or enterokinase (Invitrogen,San Diego CA) between the purification domain and the heterologousprotein can be used to facilitate purification.

The manner and method of carrying out the present invention may be morefully understood by those of skill in the art by reference to thefollowing examples, which examples are not intended in any manner tolimit the scope of the present invention or of the claims directedthereto. All references cited herein are expressly incorporated hereinin their entirety.

EXAMPLE 1

Example 1 illustrates the increase in production of subtilisin andamylase from B. subtilis having a mutation in the dciAE gene of the dciAoperon.

Production of Subtilisin from Strains Containing a dciAE Wild Type or adciAE Mutant in Shake Flasks

Strains to be tested were grown in shake flasks containing 25 ml of LB(Difco) in a 250 mL flask. Shake flasks were incubated at 37° C. withvigorous shaking and at OD 550 of 0.8, 1 mL of culture were mixed with0.5 ml 30% Glycerol and frozen for further experiments. 30 ul of thethawed vials were used to inoculate 40 ml of a media containing 68 g/LSoytone, 300 M PIPES, 20 g/L Glucose (final pH 6.8) in 250 mL flasks.The shake flasks are incubated at 37° C. with vigorous shaking for threedays, after which they are sampled for subtilisin analysis of thesupernatant.

Supernatants from liquid cultures were harvested after different timesduring growth and assayed for subtilisin as previously described(Estell, D. V., Graycar, T. P., Wells, J. A. (1985) J. Biol. Chem. 260,6518-6521) in a solution containing 0.3 mMN-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanalide (Vega Biochemicals),0.1 M Tris, pH 8.6, at 25° C. The assays measured the increase inabsorbance at 410 nm/min due to hydrolysis and release ofp-nitroanaline. Table 1 describes the yields of protease produced fromthe two strains tested.

After 24 hours the strain deleted for dciAE secreted 3.5 times moresubtilisin than the control. After 48 hours the increase is 4.9 times(Table 1).

TABLE 1 B. subtilis strains Genotype Subtilisin (rate) 48 h 2790 dciAEwt 0.888 2790 dciAE- 4.38

Production of Amylase from Strains Containing an dciAE Wild Type or andciAE Mutant in Shake Flasks

Strains to be tested were grown in shake flasks containing 25 ml of LB(Difco) in a 250 mL flask. Shake flasks were incubated at 37° C. withvigorous shaking and at OD 550 of 0.8, 1 mL of culture were mixed with0.5 ml 30% Glycerol and frozen for further experiments. 30 ul of thethawed vials were used to inoculate 40 ml of a media containing 68 g/LSoytone, 300 M PIPES, 20 g/L Glucose (final pH 6.8) in 250 mL flasks.The shake flasks are incubated at 37° C. with vigorous shaking forseveral days during which they are sampled for amylase analysis of thesupernatant.

Whole broth samples were spun down at different times of growth andtheir supernatants were assayed as follows. Supernatant is mixed in acuvete with 790.0 ul of substrate (Megazyme-Ceralpha-Alpha Amylase;substrate is diluted in water and is used as 1 part substrate plus 3parts of Alpha Amylase buffer pH 6.6) at 250 C. Alpha Amylase buffer iscomposed of 50 mM Maleate Buffer, 5 mM CaCl2, and 0.002% Triton X-100,PH=6.7. Amylase was measured in a Spectronic Genesys 2 Spectophotometerusing a protocol for amylase activity (Wavelenth: 410 nm, Initial Delay:75 secs., Total Run Time: 120 secs, Lower Limit: 0.08, Upper Limit:0.12).

Results show that the strain containing the dciAE deletion produced 1.8times more amylase at 48 hours than when the dciAE wild-type gene waspresent. At 60 hours the increase is 2.5 times (Table 2).

Table 2 describes the yields of amylase produced from the two strainstested.

TABLE 2 B. subtilis strains Genotype Amylase (rate) 48 h 60 h 2790 dciAEwt 0.054 0.066 2790 dciAE- 0.098 0.168

EXAMPLE 2

Example 2 illustrates the additional increase of production ofsubtilisin from B. subtilis having a mutation in the oppAA of the oppAoperon and a deletion of dciAA gene of the dciA operon.

Production of Recombinant Subtilisin from Strains Containing a dciAADeletion and an oppAA Mutation in Shake Flasks

Strains to be tested were grown in shake flasks containing 25 ml of LB(Difco) and 25 ug/L chloramphenicol in a 250 ml flasks. Shake flaskswere incubated at 37° C. with vigorous shaking and at OD 550 of 0.8, 1ml of culture were mixed with 0.5 ml 30% Glycerol and frozen for furtherexperiments. 25 ml of FNA34EB media (Per liter: 90.72 g PIPES acid form,pH to 6.8, 34.00 g Soytone (Difco), 2 drops of Mazu DF-204, 40 mL 50%glucose) in 250 ml flasks.

10 uL of glycerol stocks from the strains to be tested was added to eachflask. Once inoculated, the flasks were incubated at 37 degrees C., 250rpm and samples were taken every 24 hours until 75 hours. 1-mL sampleswere taken out in the sterile hood and measured for OD 550. Samples werecentrifuged for 1 minute to pellet cells.

Supernatants from liquid cultures were harvested after different timesduring growth and assayed for subtilisin as previously described(Estell, D. V., Graycar, T. P., Wells, J. A. (1985) J. Biol. Chem. 260,6518-6521) in a solution containing 0.3 mMN-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanalide (Vega Biochemicals),0.1 M Tris, pH 8.6, at 25° C. The assays measured the increase inabsorbance at 410 nm/min due to hydrolysis and release ofp-nitroanaline.

Table 3 describes the yields of protease produced from the two strains.Results show that the strain containing the dciAA deletion and the oppAAmutation produced 1.9 more protease than when the dciAA wild-type waspresent.

TABLE 3 Subtilisin (mg/L) B. subtilis strains Genotype 27 h 51 h 75 h001 oppAA- 30 105 138 040 oppAA- dciAA- 28 124 262

The effect found deleting the dciAA protein in increase enzymeproduction could be due to the possible interaction between this proteinand the subtilisin (apr) mRNA. As demonstrated in the next section weshow there is some homology between dciAA and proteins that interactwith the RNA, furthermore, we show that we found fact that there is a“putative Rnase cleavage site” in the untranslated mRNA region ofsubtilisin (starting in the +1 mRNA). This putative RnaseE cleavage sitein subtilisin has 6/7 identity with a site identified in RNA I (table4). This site is cleavaged by RNAase E (Tomcsanyi and Cohen S 1985;Lin-Chao and Cohen S 1991; Lin-Chao et al 1994). The putative Rnase Esite has 7/7 identity with the demonstrated cleavage site for RnaseE in9S RNA (Roy, M. K., and Apirion, D., Biochim. Biophys. Acta 747, 200-2081983) (Table 4).

TABLE 4 RNA location Sequence Subtilisin +1 5′ mRNA ACAGAAU (SEQ ID NO:7) RNA I +1 5′ mRNA ACAGUAU (SEQ ID NO: 8) 9S RNA +1 5′ mRNA ACAGAAU(SEQ ID NO: 9)

Without being bound to theory, we believe that it could be possible thatone of the functions of the dciAA protein is binding to this region ofthe RNA affecting the stability of this RNA or the translation of theprotein. Thus, also provided herein is a method of increasing peptideproduction/secretion of proteins that contain a putative dciAArecognition site.

Homology Between dciAA and Other Proteins

The predicted product of the dciAA gene was used to search a translationof the GeneBank data base and blast homology was found between the dciAAgene product and the dppA protein from B. methanolicus (75% identity), aputative peptide ABC transporter from Deinocuccus radiodurans (33%identity), and with the dppA dipeptide transport protein from Pyrococcusabyssi (28%). The search program used was BSORF by fasta 3 t and blast.Several dipeptide proteins from B. methanolicus 75% shows identity witha putative transport associated protein in Streptomyces coelicolor, 32%identity with a dipeptide transport protein dppA from Pyrococcus abyssi.

By doing the search using different regions of the dciAA protein in theGeneBank database (BSORF by fasta 3 t and blast) homology was foundbetween the dciAA gene product with a ribonuclease from E. coli (Rnase Eaka Ams) (Claverie-Martin et al J. Biol. Chem. 266: 2843-2851, 1991).Both proteins shows a 25% identity (and 48% conservative substitutions)over a region of 47 amino acids in the carboxy-terminal portions of bothproteins. DciAA also share 26% identity with the carboxy-terminalportion of ribonucleoprotein La from Homo sapiens (Chan et al NucleicAcid Research 17, 2233-2244,1989).

Homology Between dciAE and Other Proteins

The predicted product of the dciAE was used to search a translation ofthe GeneBank data base. The search program used was BSORF by festa 3tand blast. Homology was formed within OPPA oligopeptide-binding proteinOppA of B. subtilis (40% identity) pX02-66 of B. anthracis (32%identity), Trac of Enterococcus faecalis (30% identity), MppAperiplasmic murein peptide-binding protein precursor of E. coli (30%identity), OppA-Salty periplasmic oligopeptide-binding protein(oligopeptide-binding protein precursor) of Salmonella typhimurium (30%identity), oligopeptide permease homolog All (Borrelia burgdorferi) (29%identity), oligopeptide ABC transporter, periplasmicoligopeptide-binding protein (OppAV) homolog-lyme disease spirocheteBorrelia burgdorferi), oligopeptide-binding protein from Chlamydophilapneumonia (26% identity), and to some extent, oligopeptide ABCtransporter periplasmic binding protein OppA-syphilis spirochaete fromTreponema pallidum (26% identity).

Deletion of the Dipeptide Transport System dciAA Gene

Using the PCR technique, 647 bp of the dciAA gene present in strains 001and BG2790 was deleted. Two amplified DNA fragments containing part ofthe 5′ (using primers dci1.f and dci3.3) and 3′ of the gene (usingprimers dci2.r and dci4.0 were ligated and cloned in a pTSpUC19Kanplasmid. Plasmid pTSpUC19Kan carries a Kanamycin resistance gene (Kanr)and a temperature sensitive origin of replication (TsOri). The dciAAdisruption plasmid, pMdci, was protoplast transformed into the twostrains. Because of the TsOri, this plasmid integrated into thechromosome at the region of homology with the dciAA gene when culturedunder selective pressure at the non-permissive temperature, e.g. 48° C.After integration, strains carrying the integrated plasmid were grownextensively at permissive temperature. Upon excision of the integratedplasmid, either the parent strain 001 or BG2790 is restored, or strainscarrying the deleted gene is constructed. Two new strains were confirmedby PCR amplification of the gene with primers based in the gene sequenceto contain the deletion of the gene.

Deletion of the Dipeptide Transport System dciAE Gene

Using the PCR technique, 1228 bp of the dciAE gene present in strainBG2970 was deleted. Two amplified DNA fragments containing part of the5′ (using primers dciAE1 and dciAE2) and 3′ of the gene (using primersdciAE3 and dciAE4) were ligated and cloned in a pTSpUC19Kan plasmid.Plasmid pTSpUC19Kan carries a Kanamycin resistance gene (Kanr) and atemperature sensitive origin of replication (TsOri). The dciAEdisruption plasmid, pMdciE, was protoplast transformed into the BG2790production strain. Because of the TsOri, this plasmid integrated intothe chromosome at the region of homology with the dciAE gene whencultured under selective pressure at the non-permissive temperature,e.g. 48° C. After integration, three strains carrying the integratedplasmid were grown extensively at permissive temperature. Upon excisionof the integrated plasmid, either the parent strain BG2790 is restored,or a strain carrying the deleted gene is constructed. The new strain wasconfirmed by PCR amplification of the gene with primers based in thegene sequence to contain the deletion of the gene.

Primers:

dci1.f

GCGCGCGGATCCCGTCTGAATGAATTGTTATCGGTTTTCAGCCGTGTACGGG (SEQ ID NO: 10)

dci2.r

GCGCGCCTGCAGCGGGATGGAGATGCCGAGTACTGCAAGACTCATCGCGGCG (SEQ ID NO: 11)

dci3.r

GCGCGCGTCGACCCATATCTACTGACATGTACAATTTCATAACGC (SEQ ID NO: 12)

dci4.f

GCGCGCGTCGACGCCGCTCACACCGCCTGACAGGCCAGTTCTGAGC (SEQ ID NO: 13)

dciAE1

GCGCGCGGATCCGATGTGTCTGTCATTCTGATTACGC (SEQ ID NO: 14)

dciAE2

GCGCGCGTCGACGATCGGCGGATCGAATGMGTCGG (SEQ ID NO: 15)

dciAE3

GCGCGCGTCGACCCAATAAAGAATACGATCAGCTGATC (SEQ ID NO: 16)

dciAE4

GCGCGCCTGCAGTGTCCCAAAACCCCCGATGCGCAC (SEQ ID NO: 17)

EXAMPLE 3

Example 3 illustrates a method for screening for microorganisms with 40increased production of proteins, preferably secreted proteins. Moreparticularly, the method is for screening for cells having interruptedpeptide transport activity, particularly an inhibition in import ofpeptides, which results in an increase in production of polypeptides.The method involves subjecting cells to a toxic peptide which is thesize of a peptide which would normally be imported into the cell by thepeptide transport system. In a preferred embodiment, the toxic peptideis Bialaphos. Bialaphos is a tripeptide consisting of two L-alaninemolecules and an L-glutamic acid analogue. When digested by peptidasesit is deleterious to the cell. It is an antibiotic, see, Meiji Seika,Japan.

There has been reported in the literature that at least one operon inBacillus is involved in the uptake of molecules of the size of Bialaphos(3-amino acids), the oppA operon. There may also be other additionalgenes involved in Bialaphos uptake.

The method allows an efficient method for screening for cells which haveinterrupted peptide transport and increased production of polypeptides.Specifically, the method screens for mutation(s) which result indecreased peptide uptake. Cells which survive exposure to Bialaphos areidentified as having one or more such mutations. The method does notrequire further characterization of the mutation, for example, at thenucleic acid level, thereby providing efficiency. However, furtherscreening can occur such as for increased production of polypeptides,and further characterization and optimization of the mutation(s).Exposure to Bialaphos can occur in a number of ways such as platingcells with Bialaphos, for example, 50 ug/ml.

The interrupted peptide transport can occur because the strain ismutated, or because the cell has been mutated, or because genes,proteins or operons have been mutated or modified so as to result inincreased polypeptide production as described herein. Any one or all ofthe genes in one or more peptide transport operons may be mutated ormodified.

A microorganism or cell having a functional peptide transport operon,for example, the opp operon, will have inhibited growth. A microorganismhaving a mutation in at least one gene of the opp operon gene clusterwill not show growth inhibition in the presence of the toxicoligopeptide

Method to Obtain Bialaphos Resistant Mutants

Cells are grown in liquid culture (LB) until they reach exponentialphase. Cultures are plated onto Bacillus minimal Agar plates containingdifferent concentrations of Bialaphos. Plates are incubated overnight at37° C. and growing colonies are streaked out for purification on sametype of plates. These new strains are Bialaphos resistance and allproduce more protease than the parental strain after overnight growth.

Protease Assay

Supernatants from liquid culture were harvested after 3 days of growthand assayed for subtilisin as previously described (Estell, D. V.,Graycar, T. P., Wells, J. A. (1985) J. Biol Chem. 260:6518-6521) in asolution containing 03 mMN-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanalide (Vega Biochemicals),0.1 M Tris, pH 8.6, at 25° C. The assays measured the increase inabsorbance at 410 nm/min due to hydrolysis and release ofp-nitroanaline. The results were easily detectable and confirmed thatone can identify cells having increased protein production by screeningfor cells which do not uptake peptides.

17 1 5798 DNA Artificial Sequence dciA operon coding sequence 1ccggacgttt ttgatgaggt atttgaaaga accctgagaa aatatgaact gcttacagaa 60caggttggta aacaaacatg aatccttgaa agaggattct ttttttatca ctgaatgatt 120gagatttttc ccagttatat tgcatttttc ctcttttttt aatataattt gttagaatat 180tcataattta gtaaaaaagg aggagcgtta tgaaattgta catgtcagta gatatggaag 240gtatttcggg tcttccggac gatacctttg tggattccgg caagcggaat tatgaacgcg 300gacggcttat catgactgaa gaagcaaact actgtattgc tgaagcgttt aacagcgggt 360gtaccgaggt gctggtcaat gacagtcatt cgaagatgaa taatctgatg gttgaaaagc 420ttcaccctga agcagacttg atttctggtg acgtcaaacc attttcaatg gtggagggac 480tggatgatac gtttagaggc gctttgtttc tcggttatca tgcgagagcc tcgactcctg 540gtgtcatgtc acacagcatg attttcggcg tccgtcattt ttacataaac gatcggcctg 600tcggtgagct tggattaaat gcatacgttg ccggttatta tgatgtcccg gtattaatgg 660tagccgggga tgaccgggcg gcgaaggaag cagaagagct tatcccgaac gtgacgacag 720ccgcagtcaa acaaaccatt tcaagatccg cagtgaagtg cttgtcgcct gcgaaagccg 780gacggctgtt gacagaaaaa acgccatttg ccctgcaaaa caaggacaaa gtcaagccgc 840tcacaccgcc tgacaggcca gttctgagca ttgaattcgc caattatggc caagcagaat 900gggcgaatct gatgccggga acggaaatca agacgggaac tacaaccgtt caatttcagg 960cgaaggacat gcttgaagcc tatcaggcga tgcttgtcat gactgagctt gcgatgcgga 1020catcattctg ctaaaggggt gttttaggct ttggcgcgat acatgataaa gcgtttttgg 1080gcaatggcag ctacgatttt ggtgattacc accctgactt ttgttctcat gaaggtcatt 1140cccggatctc cttttaacga ggagagaggc acaaatgaag ccgttcaaaa aaatctcgaa 1200gcctactatc acttagacga tcctctcatt ttccaataca ttttctactt aaaatccatc 1260attacattcg atttcggacc ttcaattaaa aaaccgtcgg acagcgtaaa tgatatgctg 1320gaacgcggat ttcccgtttc ctttgagctt gggatgacag cgattgtcat tgctgtgatt 1380tctgggctgg tgctgggcgt aatcgctgca ctccgccgca atggcttttt ggactacgcc 1440gcgatgagtc ttgcagtact cggcatctcc atcccgaatt ttattctggc aacattgctc 1500attcagcaat ttgctgtcaa tctcaaacta tttcccgctg cgacatggac gagcccgatt 1560catatggtgc ttccgaccgc agcgcttgct gtagggccaa tggcgatcat tgccaggctg 1620acacggtcga gcatggtcga agttctgaca caggattata tccgcacagc aaaagcaaaa 1680gggctttctc cgttcaaaat tatcgtaaaa cacgcactca gaaatgcact catgcccgtc 1740attaccgtcc tgggcacact cgtcgccagc atcttaacag gaagctttgt cattgaaaaa 1800atctttgcca ttccgggaat gggaaaatat tttgttgaaa gcattaatca gcgggactac 1860cccgtgatta tgggaacgac cgttttttac agcgtcattc tgattatcat gctgtttttg 1920gtcgatttgg cctacggtct cttagacccg cgcattaaac tgcataagaa agggtgaagc 1980gtgtgaatct ccctgtacaa acggatgaac gccagccaga acagcacaat caggtgcctg 2040atgagtggtt tgtcttgaat caggaaaaaa atcgggaagc cgattcggtc aagcggccga 2100gtttgtcata cacgcaggat gcctggagga ggctgaaaaa aaataaatta gcgatggccg 2160gactctttat tcttttattt ctttttgtca tggcagttat cgggcccttt ttatcgcccc 2220atagtgtcgt acgccaatcg ctgacagaac aaaatcttcc gccctcagcc gatcattggt 2280tcggcaccga tgaactcggc cgggatgtgt ttacccgaac atggtatggc gcgagaatct 2340cgttgtttgt cggcgtgatg gcagcactga ttgatttttt gatcggtgtc atttacggag 2400gcgttgccgg ctataaaggc ggcaggattg acagcattat gatgcggatt atcgaagtgc 2460tgtacggact gccgtatctg cttgttgtca ttttgctgat ggtgctcatg ggaccgggac 2520tgggcacgat tattgtggcg ctgactgtga ccgggtgggt cggcatggcg agaattgtaa 2580gaggccaggt gcttcagatt aaaaattatg aatatgtact cgcctcgaaa acctttggcg 2640cgaaaacctt tcgcatcatc cggaagaatt tgctgcgcaa tactatggga gcgatcatcg 2700tacaaatgac attaaccgta cctgccgcca tattcgcaga atcattttta agctttctcg 2760gcctgggcat acaggctccg tttgcaagtt ggggcgtgat ggcgaatgac ggcctgccta 2820cgattttatc tgggcattgg tggcgcctgt tttttccggc ctttttcata tcttcgacga 2880tgtacgcgtt taatgtgctg ggggacggat tgcaggatgc gcttgaccct aagctgagga 2940ggtgactgta tggaaaaagt tctgtcagtc caaaatctgc acgtgtcttt tacgacttac 3000ggcgggacgg ttcaggcggt cagaggggtg agctttgatt tgtataaagg agaaaccttt 3060gcgatcgtcg gcgaatccgg ctgcggcaaa agcgttacct cccaaagcat catgggcctg 3120cttccgcctt attcggcgaa ggtgacagac ggcaggattc tatttaaaaa caaagacctt 3180tgccgtctct ctgacaaaga aatgagaggt ataaggggag ccgacatttc tatgattttt 3240caagacccga tgacggcgtt aaaccctacg ctgactgtcg gcgaccagct gggggaagcg 3300ctattgcgcc acaaaaaaat gagcaaaaaa gcggcacgga aagaggtgct ttccatgctg 3360tcattggttg gtattccaga tcccggagag cgcctaaagc aatatcccca ccaattcagc 3420ggcggtatga gacagcggat tgtcattgcg atggcgctga tttgcgagcc tgatatctta 3480attgcggatg aaccgaccac cgccctggat gtaaccattc aggcacagat tttagagctg 3540tttaaagaga ttcagagaaa aacggatgtg tctgtcattc tgattacgca cgatttaggg 3600gttgttgccc aggtagctga cagagtcgca gtcatgtatg ccgggaaaat ggcggaaatc 3660ggcacaagaa aagatatttt ttatcagccg cagcacccat atacaaaagg cctgctgggc 3720tctgtcccgc ggctggattt aaatggcgct gagctgaccc cgattgacgg aacgccgccg 3780gatttatttt cgcctccgcc gggctgcccg tttgccgccc gctgtccgaa caggatggtt 3840gtgtgtgaca gggtgtaccc gggccagacg atcagatctg actcgcacac cgtcaactgc 3900tggctgcagg atcaacgagc agagcatgcg gtgctgtcag gagatgcgaa ggattgaaca 3960tgaaaagggg gaagaggatg aaacgagtga aaaagctatg gggcatgggt cttgcattag 4020gactttcgtt tgcgctgatg gggtgcacag caaatgaaca ggccggaaaa gaaggcagtc 4080atgataaggc aaaaaccagc ggagaaaagg tgctgtatgt aaataatgaa aatgaaccga 4140cttcattcga tccgccgatc ggctttaata atgtgtcatg gcagccgtta aataacatca 4200tggaggggct gacgcgtctt ggcaaagatc atgagcccga gccggcaatg gcggagaaat 4260ggtctgtttc gaaagataat aaaacttaca catttacgat tcgggaaaat gcgaaatgga 4320caaacggaga tcctgtaaca gccggagact tcgaatacgc gtggaagcgg atgcttgatc 4380cgaaaaaagg cgcttcatcg gcattcctag gttattttat tgaaggcggc gaagcatata 4440acagcgggaa agggaaaaaa gacgatgtga aggtgacggc aaaggatgat cgaacccttg 4500aagttacact ggaagcaccg caaaaatatt tcctgagcgt tgtgtccaat cccgcgtatt 4560tcccggtaaa tgaaaaggtc gataaagaca atccaaagtg gtttgctgag tcggatacat 4620ttgtcggaaa cggcccgttt aagctgacgg aatggaagca tgatgacagc atcacaatgg 4680agaaaagcga cacgtattgg gataaggata cagtgaagct tgataaggtg aaatgggcga 4740tggtcagtga cagaaataca gattaccaga tgtttcaatc aggggaactt gataccgctt 4800atgtccctgc tgagctgagt gatcagctgc ttgatcagga taacgtcaat attgttgacc 4860aggcgggtct ctatttctat cgatttaatg tcaacatgga gccgttccaa aatgaaaaca 4920tcagaaaagc ctttgcgatg gctgtggatc aagaggaaat tgtaaagtac gtcacgaaaa 4980ataatgaaaa accggcgcac gcctttgtat cgcctgggtt tacgcagcct gacggcaaag 5040atttccgtga agcaggcgga gacctgatca agcctaacga aagcaaagcg aagcagctgc 5100tcgaaaaggg catgaaggaa gaaaactata ataagcttcc tgcgatcacg cttacttaca 5160gcacaaagcc ggagcataaa aagattgccg aagctattca gcaaaaattg aaaaatagcc 5220ttggagtcga tgtgaagctg gccaatatgg aatggaacgt atttttagag gatcaaaaag 5280cgctgaaatt ccaattctct caaagctcat ttttgcctga ttatgcagac cctatcagtt 5340ttctggaagc ctttcaaacg ggaaattcga tgaaccgcac aggctgggcc aataaagaat 5400acgatcagct gatcaaacag gcgaaaaacg aagccgatga aaaaacacgg ttctctctta 5460tgcatcaagc tgaagagctg ctcatcaatg aagcgccgat cattccggtt tatttttata 5520atcaggttca cctgcaaaat gaacaagtaa aaggaattgt ccgtcaccct gtcggctata 5580tcgatttaaa atgggcagat aaaaactgat ggaggcgatt gaggaaatac tgcttcttta 5640tgcgaaggag cggtattttt tctctttctt gcacgtatac gtagggtgca gagcaaatga 5700aaggagtgtt ttcgttgaat tacaagccga aagcgttgaa caagggtgat acagtcggag 5760tgatcgcgcc cgcaagtccg ccggatccaa aaaagctt 5798 2 274 PRT ArtificialSequence amino acid sequence for dciAA 2 Met Lys Leu Tyr Met Ser Val AspMet Glu Gly Ile Ser Gly Leu Pro 1 5 10 15 Asp Asp Thr Phe Val Asp SerGly Lys Arg Asn Tyr Glu Arg Gly Arg 20 25 30 Leu Ile Met Thr Glu Glu AlaAsn Tyr Cys Ile Ala Glu Ala Phe Asn 35 40 45 Ser Gly Cys Thr Glu Val LeuVal Asn Asp Ser His Ser Lys Met Asn 50 55 60 Asn Leu Met Val Glu Lys LeuHis Pro Glu Ala Asp Leu Ile Ser Gly 65 70 75 80 Asp Val Lys Pro Phe SerMet Val Glu Gly Leu Asp Asp Thr Phe Arg 85 90 95 Gly Ala Leu Phe Leu GlyTyr His Ala Arg Ala Ser Thr Pro Gly Val 100 105 110 Met Ser His Ser MetIle Phe Gly Val Arg His Phe Tyr Ile Asn Asp 115 120 125 Arg Pro Val GlyGlu Leu Gly Leu Asn Ala Tyr Val Ala Gly Tyr Tyr 130 135 140 Asp Val ProVal Leu Met Val Ala Gly Asp Asp Arg Ala Ala Lys Glu 145 150 155 160 AlaGlu Glu Leu Ile Pro Asn Val Thr Thr Ala Ala Val Lys Gln Thr 165 170 175Ile Ser Arg Ser Ala Val Lys Cys Leu Ser Pro Ala Lys Ala Gly Arg 180 185190 Leu Leu Thr Glu Lys Thr Pro Phe Ala Leu Gln Asn Lys Asp Lys Val 195200 205 Lys Pro Leu Thr Pro Pro Asp Arg Pro Val Leu Ser Ile Glu Phe Ala210 215 220 Asn Tyr Gly Gln Ala Glu Trp Ala Asn Leu Met Pro Gly Thr GluIle 225 230 235 240 Lys Thr Gly Thr Thr Thr Val Gln Phe Gln Ala Lys AspMet Leu Glu 245 250 255 Ala Tyr Gln Ala Met Leu Val Met Thr Glu Leu AlaMet Arg Thr Ser 260 265 270 Phe Cys 3 308 PRT Artificial Sequence aminoacid sequence for dciAB 3 Met Ala Arg Tyr Met Ile Lys Arg Phe Trp AlaMet Ala Ala Thr Ile 1 5 10 15 Leu Val Ile Thr Thr Leu Thr Phe Val LeuMet Lys Val Ile Pro Gly 20 25 30 Ser Pro Phe Asn Glu Glu Arg Gly Thr AsnGlu Ala Val Gln Lys Asn 35 40 45 Leu Glu Ala Tyr Tyr His Leu Asp Asp ProLeu Ile Phe Gln Tyr Ile 50 55 60 Phe Tyr Leu Lys Ser Ile Ile Thr Phe AspPhe Gly Pro Ser Ile Lys 65 70 75 80 Lys Pro Ser Asp Ser Val Asn Asp MetLeu Glu Arg Gly Phe Pro Val 85 90 95 Ser Phe Glu Leu Gly Met Thr Ala IleVal Ile Ala Val Ile Ser Gly 100 105 110 Leu Val Leu Gly Val Ile Ala AlaLeu Arg Arg Asn Gly Phe Leu Asp 115 120 125 Tyr Ala Ala Met Ser Leu AlaVal Leu Gly Ile Ser Ile Pro Asn Phe 130 135 140 Ile Leu Ala Thr Leu LeuIle Gln Gln Phe Ala Val Asn Leu Lys Leu 145 150 155 160 Phe Pro Ala AlaThr Trp Thr Ser Pro Ile His Met Val Leu Pro Thr 165 170 175 Ala Ala LeuAla Val Gly Pro Met Ala Ile Ile Ala Arg Leu Thr Arg 180 185 190 Ser SerMet Val Glu Val Leu Thr Gln Asp Tyr Ile Arg Thr Ala Lys 195 200 205 AlaLys Gly Leu Ser Pro Phe Lys Ile Ile Val Lys His Ala Leu Arg 210 215 220Asn Ala Leu Met Pro Val Ile Thr Val Leu Gly Thr Leu Val Ala Ser 225 230235 240 Ile Leu Thr Gly Ser Phe Val Ile Glu Lys Ile Phe Ala Ile Pro Gly245 250 255 Met Gly Lys Tyr Phe Val Glu Ser Ile Asn Gln Arg Asp Tyr ProVal 260 265 270 Ile Met Gly Thr Thr Val Phe Tyr Ser Val Ile Leu Ile IleMet Leu 275 280 285 Phe Leu Val Asp Leu Ala Tyr Gly Leu Leu Asp Pro ArgIle Lys Leu 290 295 300 His Lys Lys Gly 305 4 320 PRT ArtificialSequence amino acid sequence for dciAC 4 Met Asn Leu Pro Val Gln Thr AspGlu Arg Gln Pro Glu Gln His Asn 1 5 10 15 Gln Val Pro Asp Glu Trp PheVal Leu Asn Gln Glu Lys Asn Arg Glu 20 25 30 Ala Asp Ser Val Lys Arg ProSer Leu Ser Tyr Thr Gln Asp Ala Trp 35 40 45 Arg Arg Leu Lys Lys Asn LysLeu Ala Met Ala Gly Leu Phe Ile Leu 50 55 60 Leu Phe Leu Phe Val Met AlaVal Ile Gly Pro Phe Leu Ser Pro His 65 70 75 80 Ser Val Val Arg Gln SerLeu Thr Glu Gln Asn Leu Pro Pro Ser Ala 85 90 95 Asp His Trp Phe Gly ThrAsp Glu Leu Gly Arg Asp Val Phe Thr Arg 100 105 110 Thr Trp Tyr Gly AlaArg Ile Ser Leu Phe Val Gly Val Met Ala Ala 115 120 125 Leu Ile Asp PheLeu Ile Gly Val Ile Tyr Gly Gly Val Ala Gly Tyr 130 135 140 Lys Gly GlyArg Ile Asp Ser Ile Met Met Arg Ile Ile Glu Val Leu 145 150 155 160 TyrGly Leu Pro Tyr Leu Leu Val Val Ile Leu Leu Met Val Leu Met 165 170 175Gly Pro Gly Leu Gly Thr Ile Ile Val Ala Leu Thr Val Thr Gly Trp 180 185190 Val Gly Met Ala Arg Ile Val Arg Gly Gln Val Leu Gln Ile Lys Asn 195200 205 Tyr Glu Tyr Val Leu Ala Ser Lys Thr Phe Gly Ala Lys Thr Phe Arg210 215 220 Ile Ile Arg Lys Asn Leu Leu Arg Asn Thr Met Gly Ala Ile IleVal 225 230 235 240 Gln Met Thr Leu Thr Val Pro Ala Ala Ile Phe Ala GluSer Phe Leu 245 250 255 Ser Phe Leu Gly Leu Gly Ile Gln Ala Pro Phe AlaSer Trp Gly Val 260 265 270 Met Ala Asn Asp Gly Leu Pro Thr Ile Leu SerGly His Trp Trp Arg 275 280 285 Leu Phe Phe Pro Ala Phe Phe Ile Ser SerThr Met Tyr Ala Phe Asn 290 295 300 Val Leu Gly Asp Gly Leu Gln Asp AlaLeu Asp Pro Lys Leu Arg Arg 305 310 315 320 5 335 PRT ArtificialSequence amino acid sequence for dciAD 5 Met Glu Lys Val Leu Ser Val GlnAsn Leu His Val Ser Phe Thr Thr 1 5 10 15 Tyr Gly Gly Thr Val Gln AlaVal Arg Gly Val Ser Phe Asp Leu Tyr 20 25 30 Lys Gly Glu Thr Phe Ala IleVal Gly Glu Ser Gly Cys Gly Lys Ser 35 40 45 Val Thr Ser Gln Ser Ile MetGly Leu Leu Pro Pro Tyr Ser Ala Lys 50 55 60 Val Thr Asp Gly Arg Ile LeuPhe Lys Asn Lys Asp Leu Cys Arg Leu 65 70 75 80 Ser Asp Lys Glu Met ArgGly Ile Arg Gly Ala Asp Ile Ser Met Ile 85 90 95 Phe Gln Asp Pro Met ThrAla Leu Asn Pro Thr Leu Thr Val Gly Asp 100 105 110 Gln Leu Gly Glu AlaLeu Leu Arg His Lys Lys Met Ser Lys Lys Ala 115 120 125 Ala Arg Lys GluVal Leu Ser Met Leu Ser Leu Val Gly Ile Pro Asp 130 135 140 Pro Gly GluArg Leu Lys Gln Tyr Pro His Gln Phe Ser Gly Gly Met 145 150 155 160 ArgGln Arg Ile Val Ile Ala Met Ala Leu Ile Cys Glu Pro Asp Ile 165 170 175Leu Ile Ala Asp Glu Pro Thr Thr Ala Leu Asp Val Thr Ile Gln Ala 180 185190 Gln Ile Leu Glu Leu Phe Lys Glu Ile Gln Arg Lys Thr Asp Val Ser 195200 205 Val Ile Leu Ile Thr His Asp Leu Gly Val Val Ala Gln Val Ala Asp210 215 220 Arg Val Ala Val Met Tyr Ala Gly Lys Met Ala Glu Ile Gly ThrArg 225 230 235 240 Lys Asp Ile Phe Tyr Gln Pro Gln His Pro Tyr Thr LysGly Leu Leu 245 250 255 Gly Ser Val Pro Arg Leu Asp Leu Asn Gly Ala GluLeu Thr Pro Ile 260 265 270 Asp Gly Thr Pro Pro Asp Leu Phe Ser Pro ProPro Gly Cys Pro Phe 275 280 285 Ala Ala Arg Cys Pro Asn Arg Met Val ValCys Asp Arg Val Tyr Pro 290 295 300 Gly Gln Thr Ile Arg Ser Asp Ser HisThr Val Asn Cys Trp Leu Gln 305 310 315 320 Asp Gln Arg Ala Glu His AlaVal Leu Ser Gly Asp Ala Lys Asp 325 330 335 6 543 PRT ArtificialSequence amino acid sequence for dciAE 6 Met Lys Arg Val Lys Lys Leu TrpGly Met Gly Leu Ala Leu Gly Leu 1 5 10 15 Ser Phe Ala Leu Met Gly CysThr Ala Asn Glu Gln Ala Gly Lys Glu 20 25 30 Gly Ser His Asp Lys Ala LysThr Ser Gly Glu Lys Val Leu Tyr Val 35 40 45 Asn Asn Glu Asn Glu Pro ThrSer Phe Asp Pro Pro Ile Gly Phe Asn 50 55 60 Asn Val Ser Trp Gln Pro LeuAsn Asn Ile Met Glu Gly Leu Thr Arg 65 70 75 80 Leu Gly Lys Asp His GluPro Glu Pro Ala Met Ala Glu Lys Trp Ser 85 90 95 Val Ser Lys Asp Asn LysThr Tyr Thr Phe Thr Ile Arg Glu Asn Ala 100 105 110 Lys Trp Thr Asn GlyAsp Pro Val Thr Ala Gly Asp Phe Glu Tyr Ala 115 120 125 Trp Lys Arg MetLeu Asp Pro Lys Lys Gly Ala Ser Ser Ala Phe Leu 130 135 140 Gly Tyr PheIle Glu Gly Gly Glu Ala Tyr Asn Ser Gly Lys Gly Lys 145 150 155 160 LysAsp Asp Val Lys Val Thr Ala Lys Asp Asp Arg Thr Leu Glu Val 165 170 175Thr Leu Glu Ala Pro Gln Lys Tyr Phe Leu Ser Val Val Ser Asn Pro 180 185190 Ala Tyr Phe Pro Val Asn Glu Lys Val Asp Lys Asp Asn Pro Lys Trp 195200 205 Phe Ala Glu Ser Asp Thr Phe Val Gly Asn Gly Pro Phe Lys Leu Thr210 215 220 Glu Trp Lys His Asp Asp Ser Ile Thr Met Glu Lys Ser Asp ThrTyr 225 230 235 240 Trp Asp Lys Asp Thr Val Lys Leu Asp Lys Val Lys TrpAla Met Val 245 250 255 Ser Asp Arg Asn Thr Asp Tyr Gln Met Phe Gln SerGly Glu Leu Asp 260 265 270 Thr Ala Tyr Val Pro Ala Glu Leu Ser Asp GlnLeu Leu Asp Gln Asp 275 280 285 Asn Val Asn Ile Val Asp Gln Ala Gly LeuTyr Phe Tyr Arg Phe Asn 290 295 300 Val Asn Met Glu Pro Phe Gln Asn GluAsn Ile Arg Lys Ala Phe Ala 305 310 315 320 Met Ala Val Asp Gln Glu GluIle Val Lys Tyr Val Thr Lys Asn Asn 325 330 335 Glu Lys Pro Ala His AlaPhe Val Ser Pro Gly Phe Thr Gln Pro Asp 340 345 350 Gly Lys Asp Phe ArgGlu Ala Gly Gly Asp Leu Ile Lys Pro Asn Glu 355 360 365 Ser Lys Ala LysGln Leu Leu Glu Lys Gly Met Lys Glu Glu Asn Tyr 370 375 380 Asn Lys LeuPro Ala Ile Thr Leu Thr Tyr Ser Thr Lys Pro Glu His 385 390 395 400 LysLys Ile Ala Glu Ala Ile Gln Gln Lys Leu Lys Asn Ser Leu Gly 405 410 415Val Asp Val Lys Leu Ala Asn Met Glu Trp Asn Val Phe Leu Glu Asp 420 425430 Gln Lys Ala Leu Lys Phe Gln Phe Ser Gln Ser Ser Phe Leu Pro Asp 435440 445 Tyr Ala Asp Pro Ile Ser Phe Leu Glu Ala Phe Gln Thr Gly Asn Ser450 455 460 Met Asn Arg Thr Gly Trp Ala Asn Lys Glu Tyr Asp Gln Leu IleLys 465 470 475 480 Gln Ala Lys Asn Glu Ala Asp Glu Lys Thr Arg Phe SerLeu Met His 485 490 495 Gln Ala Glu Glu Leu Leu Ile Asn Glu Ala Pro IleIle Pro Val Tyr 500 505 510 Phe Tyr Asn Gln Val His Leu Gln Asn Glu GlnVal Lys Gly Ile Val 515 520 525 Arg His Pro Val Gly Tyr Ile Asp Leu LysTrp Ala Asp Lys Asn 530 535 540 7 7 RNA Artificial Sequence putativeRnase cleavage site in subtilisin 7 acagaau 7 8 7 RNA ArtificialSequence putative Rnase cleavage site in RNA I 8 acaguau 7 9 7 RNAArtificial Sequence putative Rnase cleavage site in 9S RNA 9 acagaau 710 52 DNA Artificial Sequence primer 10 gcgcgcggat cccgtctgaa tgaattgttatcggttttca gccgtgtacg gg 52 11 52 DNA Artificial Sequence primer 11gcgcgcctgc agcgggatgg agatgccgag tactgcaaga ctcatcgcgg cg 52 12 45 DNAArtificial Sequence primer 12 gcgcgcgtcg acccatatct actgacatgtacaatttcat aacgc 45 13 46 DNA Artificial Sequence primer 13 gcgcgcgtcgacgccgctca caccgcctga caggccagtt ctgagc 46 14 37 DNA Artificial Sequenceprimer 14 gcgcgcggat ccgatgtgtc tgtcattctg attacgc 37 15 36 DNAArtificial Sequence primer 15 gcgcgcgtcg acgatcggcg gatcgaatga agtcgg 3616 38 DNA Artificial Sequence primer 16 gcgcgcgtcg acccaataaa gaatacgatcagctgatc 38 17 36 DNA Artificial Sequence primer 17 gcgcgcctgcagtgtcccaa aacccccgat gcgcac 36

What is claimed is:
 1. A method of increasing secretion of a polypeptidein a cell wherein said cell is a member of the genus Bacillus thatexpresses at least one peptide transport protein comprising:inactivating at least one peptide transport protein in said cell,wherein said peptide transport protein is encoded by an operon selectedfrom the group consisting of the decoyinine inducible (dciA) operon, andthe oligonucleotide ABC transporter (opp) operon; and culturing saidcell under conditions suitable for expression and secretion of saidpolypeptide.
 2. The method of claim 1 wherein said member of the genusBacillus is selected from the group consisting of and B. licheniformls,B. lentus, B. brevus, B. stearothermophilus, B. alkalophilus, B.amylotiquefaciens, B. coagulans, B. circulans, B lautus, B.methoanolicus, B. anthracis and B. thuringiensis.
 3. The method of claim1 wherein said polypeptide is selected from the group consisting ofhormones, enzymes, growth factors, and cytokines.
 4. The method of claim1 wherein said polypeptide is heterlogous.
 5. The method of claim 1wherein said polypeptide is an enzyme selected from the group consistingof proteases, carbohydrases, reductases, lipases, isomerases,transferases, kinases, phosphatases, cellulase, endoglucosidase H,oxidases, alpha-amylases, glucoamylases, lignocellulose, hemicellulase,pectinase, and ligninase.
 6. The method of claim 1 wherein saidpolypeptide is a Bacillus amylas.
 7. The method of claim 1 wherein saidpolypeptide is subtilisin.
 8. The method of claim 1 wherein saidpolypeptide is amylase.
 9. The method of claim 8 wherein saidpolypeptide is Bacillus amylase.
 10. The method of claim 1 wherein saidinactivating comprises mutating a nucleic acid encoding said peptidetransport protein.
 11. The method of claim 10 wherein said mutatingcauses a frameshift.
 12. The method of claim 10 wherein said mutating isdeleting one or more nucleotides.
 13. A method of producing apolypeptide in a Bacillus cell comprising the steps of: a) obtaining acell comprising nucleic acid encoding a polypeptide to be produced, saidcell further comprising a peptide transport operon wherein at least onegene product of said peptide transport operon is inactive in said cell,and further wherein said peptide transport operon is selected from thegroup consisting of the decoyinine inducible (dciA) operon, and theoligonucleotide ABC transporter (opp) operon; and b) culturing said cellunder conditions suitable for expression such that said polypeptide isproduced.
 14. The method of claim 13 wherein said polypeptide isheterolgous.
 15. The method of claim 13 wherein said gene product isinactive as the result of a mutation in said operon.
 16. The method ofclaim 15 wherein said mutation is a frameshift mutation.
 17. The methodof claim 15 wherein said mutation is a